NT-proBNP was the best biomarker differentiating AF patients (area under the curve 0.995). The best NT-proBNP cut-off point to differentiate AF was 102 pg/mL; for Hs-Tn T it was 11.5 ng/L and for ST2 it was 37.7 ng/mL. It is possible that these biomarkers intervene at the onset of AF and have no role in AF maintenance.Atrial fibrillation (AF) is explained by anatomical and electrophysiological changes in the atria determined by high pressure, dilatation, infiltration and inflammation in the myocardium. There are some biomarkers implicated in these processes, namely, NT-proBNP, high sensitivity troponin (Hs-Tn), urate, galectin-3, ST2, C reactive protein and fibrinogen. The aim of this study was to assess differences in these biomarkers between patients with AF and healthy controls. We designed a cross-sectional study consecutively including all patients undergoing electrical cardioversion in our hospital for persistent AF and matched healthy controls. We included 115 patients with persistent non-valvular AF and 33 healthy subjects. The biomarkers NT-proBNP, ST2 and Hs-Tn T were significantly related to the presence of AF (1054 ± 833.30 vs. 58.31 ± 59.40,  Atrial fibrillation (AF) is the most frequent arrhythmia. It is explained by anatomical and electrophysiological changes in the atrial myocardium determined by high pressure, dilatation, infiltration and inflammation. Identification of biomarkers associated with AF may advance knowledge of AF, increasing our understanding of the pathophysiological mechanisms of the arrhythmia. Including biomarkers associated with AF in risk scales may yield predictions of AF risk with more precision in the future. Moreover, biomarkers may be used in the development of pharmacological pathways for AF preventive therapies .Some biomarkers are implicated in processes involved in AF onset and progression, namely, NT-proBNP , high sensitivity troponin T (Hs-Tn T) , urate (associated with oxidative stress), galectin-3 and ST2 (implicated in remodeling and fibrosis), C reactive protein (CRP) and fibrinogen (implicated in inflammation) .The relationship between biomarkers and AF has been previously shown , but theThe aim of this study was to assess differences in these biomarkers between AF patients and healthy subjects.This cross-sectional study included all consecutive stable patients presenting with non-valvular persistent AF, non-urgently submitted to our unit for electrical cardioversion between 17 April 2015 and 14 July 2017. We selected 1:3 aged-paired controls.An echocardiogram performed at our Cardiac Image Unit in the 6 months prior to the electrical cardioversion was required . The study was performed in accordance with the Declaration of Helsinki. Prior to cardioversion, blood samples of all patients were obtained. Blood samples of the controls were obtained on the day of inclusion. The biomarkers determined by our center’s laboratory were NT-proBNP, Hs-Tn T, galectin-3, ST2, fibrinogen, urate and CRP. Glomerular filtration rates were estimated using the CKD-EPI equation.t-test for normal quantitative variables, the U–Mann–Witney test for non-normal quantitative variables, and the Chi-square test for qualitative variables. Receiver operating characteristic (ROC) curves were obtained to assess the biomarkers’ most accurate diagnostic cut-off values. The best cut-off point corresponded to the maximum vertical distance between the ROC curve and the diagonal line. Finally, we performed multivariate analysis using logistic regression analysis including those variables that showed statistical significance (p < 0.05) in the univariate analysis.SPSS version 20.0 for Windows  was used to perform the statistical analysis. Quantitative variables were expressed as mean and standard deviation or medians, and as interquartile ranges when a normal distribution was not observed, as per the Kolmogorov–Smirnov goodness-of-fit test. Qualitative variables were expressed as frequency and percentage. In order to assess differences in biomarker levels and clinical variables in subjects with or without AF, a univariate analysis was performed using the p = 0.033).We included 115 patients with AF and 33 healthy controls. Differences in clinical characteristics between cases and controls are shown in p < 0.001 and 79.45 ± 15.23 mL/min vs. 90.54 ± 10.07 mL/min; p < 0.001, respectively), was significantly related to the presence of AF. Biomarkers NT-proBNP , ST2  and Hs-Tn T  were also significantly related to the presence of AF.Differences in analytical characteristics (including biomarkers) between cases and controls observed in the univariate analysis are shown in To assess the biomarkers’ yield, we performed ROC tests including those biomarkers that showed a significant relationship with the presence of AF in the univariate analysis. The area under the ROC curve for NT-proBNP was 0.995, for Hs-Tn T it was 0.655 and for ST2 it was 0.648. ROC curves are shown in p < 0.001) and better than ST2 (p < 0.001), but there were no significant differences between Hs-Tn T and ST2 (p = 0.99). NT-proBNP was the best biomarker for differentiating patients with AF.We used the deLong test to compare the AUCs of three biomarkers. NT-proBNP was significantly better than Hs-Tn T , for Hs-Tn T it was 11.5 ng/L (28% sensibility and 82% specificity) and for ST2 it was 37.7 ng/mL (40% sensitivity and 82% specificity).To perform multivariate analysis we included those clinical variables and biomarkers that showed a statistical significance in the univariate analysis: male sex, glomerular filtration  and biomarkers Hs-Tn T, NT-proBNP and ST2. We present the results with no dichotomized biomarkers levels, and then with dichotomized biomarkers levels, using the best cut-off points from the ROC curve.p < 0.001). In the first analysis , NT-proBp < 0.001). The odds ratio in this case was remarkably high. We found two possibilities that explain this, including the small sample size and that the vast majority of patients (97.39%) showed NT-proBNP levels above the cut-off point. As a result, in samples such as ours, the odds ratio is particularly high and should not be taken into account.In the second analysis, NT-proBNP was the only variable independently related to the presence of AF . According to previous studies, this might be due to the high prevalence of cardiovascular risk factors among men [We found a higher proportion of men in the patient group with AF than in healthy controls . Palà et al.  noted a ST2 has previously been related to AF and has been shown to have higher values in patients with persistent AF compared to patients with paroxysmal AF, which can translate progression of the AF . In contIn our study, the best ST2 cut-off point to discriminate AF was 37.7 ng/mL (40% sensitivity and 82% specificity). The association between ST2 and AF can be explained by the implication of ST2 in fibrosis and remodeling processes that initiate and maintain AF. A performance algorithm has been described in maintaining sinus rhythm based on ST2 values . This alHs-Tn T has not been classically related to AF, but it has been shown that high levels of this biomarker are associated with the incidence of AF . IncreasIn our study, the best Hs-Tn T cut-off point to discriminate AF was 11.5 n/L (28% sensitivity and 82% specificity). A relationship between Hs-Tn and AF has been previously shown. A metanalysis including 27 studies showed significantly higher Hs-Tn levels in AF patients than in subjects without AF .In a recent study that included more than 3000 patients with mild or moderate chronic kidney disease with a 7-year follow-up, Hs-Tn T values were associated with a higher risk of AF onset . AnotherIn a multicenter study of hospitalized patients with COVID-19, troponin and NT-proBNP levels were significantly higher in patients with a history of AF than in patients without a history of AF. Nevertheless, there were no differences in other biomarkers, such as CRP .Several biomarkers  did not show any relationship with the presence of AF in our sample. Our AF patients presented different AF durations, but they all had persistent AF. It is possible that these biomarkers intervene at the onset of AF and have no role in AF maintenance; it can also be explained by the small sample size.Regarding the rest of the analytical values, creatinine levels were higher in AF cases than in controls and glomerular filtration were lower in AF cases than in controls. A bidirectional relationship exists between kidney disease and cardiovascular disease (including AF) . In factOur study has some limitations. We only included patients with symptomatic persistent AF; therefore, our results cannot be extrapolated to other populations. We did not take differences in AF duration between our patients into account. We did not analyze different variables, such as previous electrical cardioversion or pulmonary veins ablation, which could impact biomarker levels and might also be affected by the presence of AF. Finally, given our study design, we cannot elucidate whether the increase in biomarker levels in our study’s sample population was a cause or a consequence of AF.On the other hand, to our knowledge this is the first study analyzing a wide battery of biomarkers in AF patients and healthy controls. The identification of pathophysiological phenomena of atrial remodeling could be useful in detecting individuals at risk of developing AF. Using biomarkers to detect AF risk could facilitate the application of more exhaustive diagnostic procedures for affected patients.In our sample, NT-proBNP, ST2 and Hs-Tn T were related to the presence of AF. NT-proBNP showed the highest yield in differentiating patients with AF from healthy subjects, and was the only biomarker independently related to the presence of AF.


From the advantage of the cross-linked structures made with the use of polymeric cross-linkers, a stable membrane can be obtained even though the polymer matrix with a very high degree of sulfonation was used. In particular, hydrophilic cross-linker is found to be effective in improving physicochemical properties of the cross-linked membranes and at the same time showing reasonable proton conductivity. Accordingly, membrane electrode assembly made from the cross-linked membrane prepared by using hydrophilic polymeric cross-linker exhibits outstanding cell performance under high temperature and low relative humidity conditions (e.g., maximum power density of 176.4 mW cm Th. Th25]. 4 as the reagent and catalyst  was added into a 250 mL two-neck, round-bottom flask equipped with a condenser and a magnetic stirrer maintained under N2 atmosphere. Then, 4.138 g (15.10 mmol) of SnCl4 and 1.939 g (22.63 mmol) of chloromethyl methyl ether were added to the reactor at 25 °C. The solution mixture was heated at 50 °C and stirred for 24 h. The mixture was then poured into excess isopropyl alcohol (IPA). The precipitate was filtered and rinsed several times with methanol and deionized (DI) water. After drying at 60 °C in a vacuum oven for 12 h, SPAES-Cl was obtained (yield = 85%). As confirmed through 1H-NMR, the concentration of the chloromethyl groups in SPAES-Cl was approximately 6 mol%, indicating that 0.06 equivalents of the chloromethyl groups were introduced into the repeating unit.SPAES was modified to SPAES-Cl through a substitution reaction using chloromethyl methyl ether and SnClcatalyst , respect2 gas inlet and outlet, and stirring bar. Then, a mixture containing 3.00 g (11.98 mmol) of TBBT, 4.71 g (9.58 mmol) of SDCDPS, and 2.65 g (19.17 mmol) of K2CO3 in 28 mL of N-methyl-2-pyrrolidone (NMP) was added into the flask. Afterwards, 14 mL of toluene  was added as an azeotropic agent. To ensure complete dehydration, the reaction mixture was heated to 145 °C for 4 h. After toluene was completely removed, the temperature was increased to 160 °C, and the reaction was continued for another 4 h. After the reaction, the solution was cooled to room temperature and then filtered to remove the salt produced during the polymerization. The homogeneous solution was then poured into IPA to precipitate the polymer, which was filtered, rinsed several times with methanol and IPA alcohol, and dried in a vacuum oven for 12 h at 60 °C. The product polymer yield was 80%.Polymeric cross-linkers with different hydrophilicity  were synthesized through the condensation polymerization of dithiol monomers and dichloro monomers . ThiopheThiophenoxide-terminated poly(arylene thioether sulfone) (DT) was also prepared through a condensation polymerization reaction using 3.00 g (11.98 mmol) of TBBT and 2.75 g (9.58 mmol) of DCDPS. Unlike in the preparation of SDT, the reaction mixture for DT synthesis was not subjected to a dehydration process using toluene due to the high reactivity of DT and DCDPS. The homogeneous solution obtained after a reaction at 160 °C for 4 h was poured into methanol and then rinsed with DI water several times. After drying at 60 °C in a vacuum oven for 12 h, DT was obtained at a yield of 91%.The cross-linked SPAES membranes were prepared through the in-situ casting and heating of SPAES-Cl solutions with different cross-linkers, such as SDT and DT. The cross-linked membranes containing SDT and DT were designated as SDT-CSPAES and DT-CSPAES, respectively. SDT-CSPAES membrane was prepared by dissolving 0.6000 g (1.1261 mmol of repeat units) of SPAES-Cl, 0.0716 g (0.04447 mmol) of SDT, and 0.1160 g (1.1261 mmol) of triethylamine (TEA) in 4.400 mL DMSO. An appropriate amount of SDT was added to ensure that the molar ratio between the potassium thiophenoxide end-group of SDT and the chloromethyl groups of SPAES-Cl was 1:1. The amount of TEA was determined based on the molar content of the potassium thiophenoxide end-group of SDT. Lastly, the amount of DMAc was adjusted to obtain an approximate total concentration of SPAES-Cl, SDT, and TEA of 12 wt%. The solution mixture was spread onto a clean glass plate. The thickness of the film was controlled using a doctor blade. The cast solution was heated to 120 °C in a vacuum oven for 12 h. During the heat treatment, the cross-linked membrane was formed through solvent evaporation and a simultaneous nucleophilic substitution reaction between the chloromethyl groups and potassium thiophenoxide groups. To detach the synthesized film and remove the remaining solvents and TEA, the coated glass plate was soaked in and washed with DI water several times. DT-CSPAES membrane was fabricated through a similar preparation method; however, for this case, DT and DMAc were used as cross-linker and solvent, respectively. After drying in a vacuum oven at 60 °C for 24 h, cross-linked membranes with an approximate thickness of 15–20 µm were obtained.For comparison, a linear SPAES membrane was prepared by casting a 12 wt% SPAES solution in DMAc onto clean glass. The employed film preparation method, which included solution casting, heat treatment, and drying, was similar to that used to prepare the cross-linked SPAES membranes. The thickness of the synthesized linear SPAES membrane was approximately 25 µm.TM ionomer dispersion  and a water–n-propanol solvent mixture. To ensure the uniformity of the catalyst ink, the dispersion was mixed using an ultrasonic vibrator . Then, a catalyst layer was developed by coating the ink mixture onto a PTFE sheet, which was subsequently dried at 60 °C. The MEA was fabricated by hot pressing the catalyst layer and the membrane together, and then transferring the catalyst layer onto the membrane at 120 °C. On one hand, the Pt loading at the MEA was 0.4 mg/cm2. On the other hand, the ionomer content in the catalyst layer was approximately 30%.MEAs made from SDT-CSPAES membrane and recast-Nafion were prepared through a typical decal method ,26. Cata1H-NMR spectra were recorded using an Avance-400 spectrometer  with a proton frequency of 400 MHz. Deuterated dimethylsulfoxide and tetramethylsilane were used as the solvent and internal standard, respectively. The Fourier-transform infrared (FT-IR) spectra of the films were recorded using a Cary 660 FT-IR spectrometer  in the attenuated total reflectance mode at ambient temperature. Data were collected from 32 scans at a 4 cm−1 resolution. To avoid differences caused by the pressure and penetration depth, the sample was placed in equal physical contact with the sampling plate of the spectrometer accessory.2) was measured using a fuel cell test station  at ambient pressure and 120 °C. The flow rates of H2 at the anode and air at the cathode were 50 and 200 cm3 min−1, respectively. The inlet gases were humidified to 40% relative humidity (RH). Voltage values at each current density were recorded after 1 min of current application [For the full cell testing, cells were assembled by placing two gas diffusion layers  on both sides of the MEA. The performance of each cell , water uptake, area-based swelling ratio, and proton conductivity, are described in the 1H-NMR spectra of SPAES and SPAES-Cl. From the 1H-NMR analysis, the DS of SPAES (66 mol%) was smaller than the expected value, possibly due to the reactivity of the sulfonated dichloro monomer being lower than that of the unsulfonated dichloro monomer [SPAES was synthesized through condensation polymerization.  monomer . To intr1H-NMR and FT-IR analyses were carried out  were synthesized as polymeric cross-linkers . Given tried out a,b. The ectively ,29,30.Transparent and flexible cross-linked SPAES membranes were prepared through the in situ casting and heating of a DMAc solution containing SPAES-Cl and cross-linkers (SDT or DT) with a small amount of a liquid base catalyst (TEA) . The croTo assess membrane stability, the water-absorbing ability and swelling behavior of the cross-linked membranes were compared to those of the linear SPAES membrane. −2, respectively. Similar to previous findings, the high proton conductivity of SDT-CSPAES membranes can be ascribed to the high hydrophilicity of SDT, which allowed the SDT-CSPAES membranes to absorb water more efficiently than the DT-CSPAES membranes. Since SDT has two sulfonic acid groups per repeat unit, the number of sulfonic acid groups in the membrane after cross-linking would increase. This results in a relatively larger water uptake and forms large hydrophilic domains that may facilitate proton transfer, resulting in high proton conductivity even under very low RH conditions [−1) between the SDT-CSPAES and DT-CSPAES membranes, indicating the presence of different numbers of ion exchangeable groups per unit mass of the dry membrane, also supported the conductivity difference of these two membranes.nditions . As a re−2) than that made from the recast-Nafion (169.0 mW cm−2). The expected cell performance improvement corresponding to the high proton conductivity of the SDT-CSPAES membrane was not achieved because the MEA preparation processes and components were optimized for the PFSA ionomer membranes. Nevertheless, the results demonstrate that the SDT-CSPAES membrane prepared with use of a highly sulfonated polymer and hydrophilic polymeric cross-linker was an effective cross-linked membrane for PEMFC applications.Based on the measured mechanical properties and proton conductivity, the SDT-CSPAES membrane was used for the fuel cell performance evaluation. Since the cell performance of linear SPAES membrane cannot be measured due to the failure of the membrane in the activation step, recast-Nafion (20–25 μm) was used as a reference membrane for comparison. −2 than that of the MEA made from recast-Nafion. Therefore, we concluded that the cross-linked membranes made from highly hydrophilic polymeric cross-linkers via a facile casting process are applicable to various PEM materials prepared using highly sulfonated polymers for practical PEMFC applications. In addition, since the construction of the cross-linked structure of the CSPAES membrane system is easily tuned by changing the type of polymeric cross-linkers and the modification degree of the SPAES main chain, this cross-linking strategy could apply in various polymer electrolytes used in energy devices.In this study, cross-linked SPAES membranes were synthesized using polymeric cross-linkers through a facile in situ casting method. With the aid of polymeric cross-linkers, the cross-linked membranes exhibited much improved physicochemical properties, including dimensional stability and mechanical strength, given the high degree of sulfonation (DS) value of SPAES. Additionally, the effects of the hydrophilicity of the cross-linkers on the stability and proton conductivity of the cross-linked membranes were studied. The water absorption ability of the cross-linked membrane prepared using the hydrophilic SDT cross-linkers increased without a considerable reduction in membrane stability; thus, the membrane exhibited relatively high proton conductivity at high temperature and low RH. As a result, the cell performance of the MEA made from this membrane is even higher with the maximum power density of 176.4 mW cm


Data from the “Trattamento Ormonale nello Scompenso CArdiaco” (T.O.S.CA) registry showed that heart failure (HF) represents a complex clinical syndrome with different hormonal alterations. Renal failure represents a frequent complication in HF. We evaluated the relationship between renal function and insuline-like growth factor-1 (IGF-1) deficiency and its impact on cardiovascular mortality (CVM) in patients enrolled in the T.O.S.CA. registry.At the enrolment, all subjects underwent chemistry examinations, including circulating hormones and cardiovascular functional tests. COX regression analysis was used to evaluate factors related to CVM during the follow-up period in all populations, in high-risk patients and in the young-adult population. Also, we evaluate the effects of renal function on the CVM.337 patients (41 deceased) were analyzed. CVM was related to severe renal dysfunction (HR stages IV–V = 4.86), high-risk conditions (HR 2.25), serum IGF-1 (HR 0.42), and HF etiology . In high-risk patients, CVM was related to IGF-1 levels, severe renal dysfunction and valvular etiology, whereas in young patients CMV was related to the high-risk pattern and serum IGF-1 levels.Our study showed the clinical and prognostic utility of the IGF-1 assay in patients with HF.The online version contains supplementary material available at 10.1007/s11739-022-02980-4. Heart failure (HF) represents an important public health problem, affecting around 23 million patients worldwide . The HF The HF is a complex clinical syndrome affecting different functional systems of the body. Although the causes leading to HF are mainly related to cardiac alterations, HF is undoubtedly associated with multi-organ dysfunctions affecting lungs, kidneys, brain and endocrine glands. Notably, a multihormonal deficit syndrome , characterized by changes in the catabolism/anabolism balance, has been reported in patients with HF , 4. HormRenal failure is a well-known risk factor for further cardiovascular diseases in patients with HF  and, at The aim of this study was to evaluate whether a relationship between renal function and IGF-1 deficiency exist and its impact on cardiovascular mortality in patients with moderately or severely HFrEF, enrolled in the T.O.S.CA. registry.Data for the present analysis come from the T.O.S.CA. Registry, an out-come-oriented, multicenter, prospective observational study, designed to evaluate the prevalence of multiple hormone deficiency syndromes in patients with CHF and its impact on clinical outcomes. The study design has been already published . BrieflyAfter enrolment, all subjects were followed as outpatients with each procedure and blood sample collection that were repeated annually during the study period. Intermediate visits were scheduled for clinical follow-up, therapy monitoring, and to collect relevant clinical event information .http://www.registrotosca.com).Clinical data and index events from enrolled patients identified by a unique identifier number were updated on a Web-based platform  mortality, a COX regression analysis was performed including age, sex, chronic kidney disease stage calculated using the CKD-EPI formula [Data are shown as absolute numbers, percentages, and means ± standard deviation (SD). A descriptive analysis of the whole population was performed, followed by a comparison of survivors and deceased during the follow-up period. The analysis of variables was performed by using the Chi-Squared test, the Student  formula , logaritA total of 337 patients  with a mean age of 63 ± 12 years were analyzed.2. Most patients (66.5%) had normal or mildly decreased renal function  [The analysis of renal function showed an average eGFR of 72.9 ± 25.4 ml/min/1.73 m1.73 m2) .After a mean follow-up period of 44 ± 11 months, the percentage of deaths was 12.2% (41 patients); the clinical and laboratory characteristics of the cohort stratify by living status are reported in Table p = 0.003), with a more advanced NYHA class and ventricular dysfunction based on EF values , with a higher serum BNP , with a reduced renal function  and with lower serum IGF-1 values . No differences were present according to treatment. HF secondary to ischemic heart disease (IHD) and valve diseases were the 2 etiologies more likely associated with cardiovascular mortality , , respectively; (ii) being classified as high-risk patient of mortality/unexpected death , and (iii) serum IGF-1 . The underlying valvular etiology of cardiomyopathy was strongly associated with CV mortality , while the HR concerning the ischemic form was 1.63 .COX regression analysis Table  showed tFigure In high-risk patients it was possible to highlight how the factors associated with higher mortality were IGF-1 values, the presence of severe renal dysfunction and the valvular etiology Table A. AnalyzThe main findings of the present analyses are that in a cohort of HFrEF the serum IGF-1 level independently predicted CV mortality, more so in younger patients and regardless of HF severity and renal function.Prior work showed that the serum IGF-1 was a predictor of mortality from cardiovascular events in patients with HFrEF . Our finp = 0.001) and the changes in the NYHA class . Beneficial effects of exogenous administration of GH were also highlighted in the case of shorter treatments (up to 6 months) in a cohort of 28 patients enrolled in the T.O.S.CA. registry [Evaluation of IGF-1 is important because this hormone can be considered as a modifiable factor. Previous studies showed that exogenous administration of GH in patients with HF induces an improvement in functional and instrumental performances. Indeed, the study conducted by Cittadini et al. on a cohort of 17 patients treated with GH (compared with 14 control patients)  showed nSimilar effects on EF were also shown in an Iranian study for shorter period of GH administration (up to 3 months) in patients with post-myocardial infarction HF and reduced EF (8 treated patients compared with 8 controls). However, the beneficial effect tended to decrease after about 9 months from the interruption of the therapy .This indirectly indicates the clinical and functional importance of IGF-1 in patients with HF and the beneficial and protective effects by this hormone towards physical performances in a highly prevalent disease such as HF itself.At the same time, it is also important to underline how a meta-analysis conducted in 2007 showed how the beneficial effects of GH administration in amelioration of EF (+ 5.1%), diameter of the left ventricle (− 2.02 mm), NYHA class (− 0.97) and duration of physical exercise (+ 103.7 s) were influenced by sex, being more favorable in males than females . The cauCittadini et al.  demonstrOur results did not show any relationship between serum IGF-1 and EF or with the degree of renal impairment, confirming that serum IGF-1 would not seem to be influenced by the presence of such abnormalities. These findings are in agreement with the study of Ravassa et al. on a group of 686 patients with HF. These authors showed no relationship between IGF-1 and renal dysfunction, while a significant difference was found in circulating protein binding insulin-like growth factor 2 (IGFBP-2), whose levels are usually higher in patients with impaired renal function . It is kNeprilysin inhibitors have been recently introduced in the management of patients with HF and concomitant renal insufficiency levels , with a This study has several limitations: first of all its retrospective nature. Moreover, our analyses did not take into account patients’ comorbidities that could increase mortality in subjects with HF. It has to be considered that comorbidities, such as diabetes mellitus and CKD have a negative prognostic effect on patients with HF,consequent to alterations in the serum levels of IGF-1. Furthermore, none of the patients were under sacubitril/valsartan therapy. This was because this treatment was not recommended by current guidelines at the time of enrollment. Moreover, this drug cost is refundable in Italy only starting from 2018.Serum levels of IGF-1 may also be affected by pulsatile GH secretion. In healthy patients, GH is secreted according to a complex circadian rhythm and with different secretory peaks between males and females in terms of both the time of onset and the duration of the secretory peaks themselves , 40. ForIn addition, some drugs normally used in clinical practice, such as ACE inhibitors, could induce a modification in the serum concentrations of IGF-1 and its binding proteins, with a more significant effect in patients with HFrEF , 41. ThAmong the conditions that could modify the serum concentrations of IGF-1, we could also mention the association between cardiac decompensation and hepatocellular dysfunction, having cirrhosis a negative effect on IGF-1 peripheral secretion , 44. FinOur study demonstrates both the clinical and prognostic utility of the IGF-1 assay in patients with HFrEF. In particular, in patients at higher cardiovascular risk, the evaluation of the GH/IGF-1 axis could be useful in predicting the prognosis and planning the therapeutic strategies. This kind of approach could represent useful clinical stratification of prognosis in patients with HF.We believe that patients with HFrEF, would benefit from early detection of GH/IGF-1 alterations, in order to adequately predict their prognosis. At the same time, clinicians could eventually plan correction of hormonal alterations, with the aim of improving long-term outcomes.Supplementary file1 (DOCX 128 KB)Below is the link to the electronic supplementary material.


Alzheimer's disease (AD) is a neurodegenerative condition that is pathologically characterized by the presence of amyloid plaques and neurofibrillary tangles. Animal models of AD have been useful in understanding the disease process and in investigating the effects of compounds on pathology and behavior. APP/PS1 mice develop amyloid plaques and show memory impairment. Cyclic glycine-proline (cGP) is a cyclic dipeptide that is likely produced from a tripeptide, glycine-proline-glutamate, which itself is generated after proteolytic cleavage of insulin-like growth factor-1. Here, we show that cGP improves spatial memory and reduces amyloid plaque burden in APP/PS1 mice. The results thus suggest that cGP could potentially provide beneficial effects in AD. The Aβ peptide is produced by the proteolytic processing of amyloid precursor protein (APP) [β causes oxidative stress, neuroinflammation, and toxicity to neurons [β causes synaptic dysfunction and deficits in synaptic plasticity and memory [Alzheimer's disease (AD) accounts for majority of the dementia cases in the elderly population. Accumulation of misfolded proteins is a key neuropathological feature of AD. Intracellularly, neurofibrillary tangles containing hyperphosphorylated tau and, extracellularly, amyloid plaques are found in AD brains. These deposits cause cellular dysfunction and eventually cell death. The widely accepted hypothesis of AD, the amyloid cascade hypothesis , posits in (APP) , and it  neurons –6. Impord memory , 7–9.Transgenic animals are widely used to model AD and study its pathology and behavioral deficits. The double transgenic mice with mutations in APP and presenilin-1 (PS1), commonly known as APP/PS1 mice, are widely used animal model for AD. These mice develop amyloid plaques and show impairment in memory .β burden [Insulin-like growth factor-1 (IGF-1) plays crucial roles during brain development . Intranaβ burden .IGF-1 is processed into an N-terminal tripeptide, glycine-proline-glutamate (GPE), and des-N-(1-3)-IGF-1 , 19. CycThe double transgenic mice, B6;C3-Tg85Dbo/Mmjax (APP/PS1), originally obtained from The Jackson Laboratory, were bred in the animal facility of National Brain Research Centre. These mice overexpress a chimeric mouse/human APP (Mo/HuAPP695swe) and a mutant human presenilin-1 (PS1-dE9) under a prion promoter.ad libitum. The procedures were approved by the Institutional Animal Ethics Committee.Mice from the colony were tested for the presence of the transgenes using the APP and the PS1 primers . Nine- t2HPO4, 1.76 mM KH2PO4, and pH 7.4) at a concentration of 80 μg/μl and administered intranasally at a dose of 20 mg/kg body weight. Taking clues from previous studies that used a dipeptide analogue of piracetam [The animals were randomly distributed into the control and the experimental groups to avoid any bias. Each animal was handled for 5-10 min for 5 days. cGP (Cyclo(-Gly-Pro); Bachem, cat. no. G-1720) was dissolved in phosphate-buffered saline  task, which has been extensively employed in the literature, to study spatial memory. It has been shown that the APP/PS1 mice show deficit in spatial memory , 25.The task was carried out in a circular pool, 1.68 m in diameter and 60 cm in height. The pool was half-filled with water, and the water temperature was kept at 24 ± 2°C. A circular platform  was placed 1 cm below the water surface. To hide the platform, a nontoxic white paint was mixed in water to make it opaque. At a distance of 40-60 cm around the pool, four objects  were put as spatial cues. One-hundred-watt electric bulbs facing the roof were placed at the four corners of the room to keep the pool illuminated. The positions of the platform in the pool and the spatial cues around the pool were kept constant throughout the training phase. The platform was removed during the testing period while the spatial cues were retained at their original places.WT mice and APP/PS1 mice were trained in MWM task using a modified version of the paradigm used previously . The traOnce gently released, the animals were allowed to freely explore the pool for 2 min and find the hidden platform. If they failed to find the platform within the stipulated time, they were manually directed to the platform and placed on it. The mice stayed on the platform for 30 seconds. The release positions were pseudorandomized each day. Escape latency was calculated as the time taken by the animals to reach the platform after their release in the water. The escape latency values in the 4 trials each day were averaged to get a single value for each animal on that day.The testing  was conducted 24 h after the last training session to assess memory. In probe trial, each animal was given 2 min to explore the pool. The spatial memory was assessed using the parameters, escape latency (time taken to reach the former platform area), number of times the animals crossed the annulus (former platform area), and time spent in the platform quadrant (the quadrant which housed the platform during the training).To record the behavior of the animals during training and testing phases, a webcam (Logitech) was installed above the pool. The path followed by the animals in the pool was tracked using ANYMAZE tracking software (ANYMAZE). The videos were collected in a hard disk and analyzed manually.th day. The animals were anaesthetized with halothane and decapitated. The brains were taken out and washed with ice-cold PBS. The two hemispheres of the brain were separated. The left hemisphere of the brain from half of the animals and the right hemisphere of the brain from other half of the animals were taken and fixed overnight in 4% paraformaldehyde. In the next day, the samples were transferred to a 20% sucrose solution in PBS until they sank to the bottom, after which they were transferred to a 30% sucrose solution in PBS until they sank to the bottom.A separate set of mice was used for these experiments. WT and APP/PS1 mice received cGP intranasally for 28 days. They were sacrificed 4-5 h after receiving the drug on the 28μm thick sections using cryostat (Leica CM3050 S) and placed in the wells of 24-well cell culture plates containing PBS with 0.01% sodium azide. Every 6th section of a hemisphere was mounted on a slide, with a total of 5 sections per hemisphere. A single slide contained mounted sections from all the experimental groups.The samples were sectioned into 40 β-sheets. The sections were stained with thioflavin-S to visualize amyloid plaques [2), percentage area covered by plaques, size of remaining plaques, and total area defined for analysis. Raw data values from all the five sections of a particular hemisphere were averaged to get a single value for each animal. Only in the representative images shown in the figures, for clarity, the brightness and contrast values were adjusted identically in both groups.Thioflavin-S  is a benzothiazole dye that displays enhanced fluorescence on binding to fibrillar  plaques . The stat-test (two-tailed). The group differences were considered significant when p value was < 0.05. Data are presented as mean ± SEM.The data presented in p < 0.01). Importantly, this deficit in acquisition was alleviated by treatment with cGP, as there was a significant difference in latency between untreated APP/PS1 mice and cGP-treated APP/PS1 mice . The cGP-treated WT mice did not differ from WT mice indicating comparable learning in these two groups of mice .Several studies have shown that APP/PS1 mice are impaired in spatial memory , 25. We p < 0.01; p < 0.01; p < 0.01). Importantly, the deficit in LTM observed in APP/PS1 mice was alleviated by treatment with cGP. There were significant differences in the three parameters between untreated APP/PS1 mice and cGP-treated APP/PS1 mice . cGP-treated WT mice did not differ in these parameters from WT mice indicating comparable LTM in these two groups of mice . During the LTM test, the three parameters used to assess spatial memory were escape latency, time spent in the platform quadrant, and number of annulus crossings. Significant differences in all the three parameters were found between WT and APP/PS1 mice showing that APP/PS1 mice were deficient in LTM . The average size of remaining plaques in cGP-treated APP/PS1 mice was similar to plaque size in untreated APP/PS1 mice . The total area used for analysis was comparable between APP/PS1 mice and cGP-APP/PS1 mice . The results presented here show that cGP reduces amyloid plaque load in the hippocampus of APP/PS1 mice.In the hippocampus, no amyloid plaques were observed in WT mice and cGP-WT mice. The representative images presented in 2, APP/PS1, 42.7 ± 7.7; cGP-APP/PS1, 15.4 ± 1.2, p < 0.02; p < 0.04). The average size of remaining plaques in the cGP-treated APP/PS1 mice was comparable to plaque size in the untreated APP/PS1 mice . The results are similar to those observed in the hippocampus after cGP treatment of APP/PS1 mice. Collectively, the results show that cGP reduces amyloid pathology in two brain regions, i.e., the hippocampus and cortex of APP/PS1 mice.The effect of cGP on amyloid pathology in the cortex of the same mice was also assessed. Similar to the hippocampus, no amyloid plaques were observed in WT mice and cGP-WT mice in the cortex. The representative images in β deposition in the brain [Alzheimer's disease is the most common cause of dementia in the geriatric population. Transgenic animals have been widely used to study the development of AD. These animal models show impairments in long-term potentiation and long-term depression . Mice ovhe brain . Althoughe brain , 25, theIGF-1 is essential for development . In addicGP and its analogue (NNZ 2591) have been shown to be neuroprotective in hypoxic-ischemic injury with NNZ 2591 even improving sensory-motor function . Furtherβ- and mutant APP-induced toxicity [β load in APP/PS2 mice [The mechanisms involved in the effects of cGP on memory and amyloid plaque load are not known. cGP regulates IGF-1 homeostasis possibly by regulating its binding to the binding protein. cGP promotes IGF-1 activity when it is insufficient and inhibits IGF-1 activity when it is in excess . Althougtoxicity , 37. Inctoxicity , and IGFPS2 mice . ConsideWe have not determined the presence of cGP in the brain after intranasal administration. Previous studies have shown the presence of subcutaneously injected NNZ 2591 or intraperitoneally injected GPE in the cerebrospinal fluid , 39. ThuA small cyclic peptide, cGP, endogenously generated from IGF-1, is shown to improve memory in APP/PS1 mice. This peptide reduces plaque load in the hippocampus and cortex of APP/PS1 mice. Hence, this could prove beneficial in mitigating AD pathology.


Beta-carotene (BC) is a well-known antioxidant. However, increasing evidence shows that under severe oxidative conditions, BC can become pro-oxidant, an effect that may be enhanced in the presence of iron (II). In our earlier studies, we observed that despite increasing heme oxygenase-1 (HO-1) levels in the heart, the protective effects of BC have been lost when it was used at a high concentration. Since iron releases from heme as a consequence of HO-1 activity, we hypothesized that the application of an iron-chelator (IC) would reverse the lost cardiac protection associated with an elevated HO-1 level. Thus, in the present study, we investigated the effects of desferrioxiamine (DFO) in isolated, ischemic/reperfused rat hearts after long-term treatment with vehicle or high-dose (HD) BC. Vehicle or 150 mg/bw kg daily doses of BC were administered to the rats for 4 weeks, and then their hearts were removed and subjected to 30 min of global ischemia (ISA) followed by 120 min of reperfusion (REP). During the experiments, cardiac function was registered, and at the end of the REP period, infarct size (IS) and HO-1 expression were measured. The results show that DFO treatment alone during REP significantly ameliorated postischemic cardiac function and decreased IS, although HO-1 expression was not increased significantly. In hearts isolated from BC-treated rats, no cardioprotective effects, despite an elevated HO-1 level, were observed, while DFO administration after ISA resulted in a mild improvement in heart function and IS. Our results suggest that iron could have a role whether BC exerts antioxidant or pro-oxidant effects in ISA/REP-injured hearts. Despite intensive research worldwide, there is a constant increase in the number of patients suffering from any kind of oxidative stress- (OS) related disease . The imb2+ from heme as a consequence of the HO-1 activity. Furthermore, it was shown that iron has a key role in myocardial ISA/REP-induced damage [2+ enhances the formation of various BC cleavage products (CPs), which suggests that increased HO-1 activity in the presence of high BC levels potentially overwhelms the cytoprotective effects of HO-1 and exacerbates OS on the myocardial tissue.The detrimental effects of OS can be reduced by the administration of exogenous antioxidants. The most favored exogenous antioxidants are flavonoids, ascorbic acid, vitamin E and carotenoids, including BC. Although BC has well-documented antioxidant properties and health effects , a numbed damage ,22,23. Td damage ,24, as wd damage ,25. One d damage . Howeverd damage  and ferrd damage . Furtherd damage  showed t2+ that participates in the Fenton reaction, thus preventing the formation of hydroxyl radicals and subsequent oxidative stress [If iron overload associated with elevated HO-1 expression contributes at least partly to the diminished cardiac protection of a high BC dosage, then the elimination of excess iron from the tissue could abolish the earlier observed loss of protection. Thus, in the present study, we investigated the effects of DFO, an IC, which chelates the Fee stress  on postip < 0.05), as well as in the case of BC-treated and DFO-administered hearts after 30 min of REP (* p < 0.05), but not in the case of hearts isolated from rats, which received BC treatment alone (p < 0.05) (p < 0.05), but no differences were detected in the case of hearts isolated from BC-treated animals (p < 0.05)  B. An eva < 0.05) C reveale animals C. Treatm animals D. Likewi < 0.05) E.p < 0.05) (The extent of the effect of DFO treatment on the infarcted area in ISA/REP-injured hearts is shown in  < 0.05) -DFO. Thi < 0.05) -BC. It w < 0.05) -BC + DFOp < 0.05) irrespective of the administration of DFO  and the “Guide for the Care and Use of Laboratory Animals”. Maintenance and treatment of rats used in the present study were additionally approved by the Institutional Animal Care and Use Committee of the University of Debrecen, Debrecen, Hungary (6/2019/DEMAB).The experiments were accomplished using adult male Sprague-Dawley rats (Charles River Laboratories) with a body weight range of 350–400 g. The animals were housed in wire-bottomed cages (three rats per cage) throughout the study (4 weeks) and provided with normal laboratory rodent chow pellets  and water n = 10 in each group). Two groups received hydroxyethyl cellulose-water (1:4) while animals in the remaining two groups were treated with BC (150 mg/kg/day) suspended in hydroxyethyl cellulose-water (1:4) by gavage daily for 4 weeks, respectively. All-trans-BC was obtained from Merck Kft. . Following the 4-week treatment with vehicle or BC, the rats were anesthetized with an intraperitoneal ketamine-xylazine injection (75/10 mg/kg), and intravenous heparin was given as anticoagulant (1000 IU/kg). Following the induction of deep anesthesia, chest cavities were opened, and hearts were excised and placed in ice-cold modified Krebs-Henseleit bicarbonate (KHB) buffer to prevent damage to cardiac tissue. After excision of the hearts, aortas were cannulated, and hearts were perfused with modified KHB buffer at a filling pressure of 100 cm of water using the Langendorff (non-working) mode of the isolated working heart setup for 5 min to flush blood out from the hearts. During the washout period, pulmonary veins were cannulated, and heart function was assessed in working mode at a filling pressure of 17 cm of water with KHB buffer [Rats were segregated into 4 groups (Hearts obtained from rats receiving vehicle were further divided into two groups. The first group was used as control (C), and hearts in the other group were treated with DFO (100 μM) during the REP period of the ex vivo experiments. Similarly, hearts isolated from BC-treated rats were also further divided into two groups, and the first group was not treated with DFO (BC), while hearts in the other group received DFO (100 μM) treatment (BC + DFO) during REP of the isolated heart experiments. DFO was dissolved in the perfusion buffer.Following 10 min of aerobic working perfusion, 30 min of global ISA was initiated by clamping the pulmonary inflow and the aortic outflow. At the end of the ischemic period, 120 min of REP was started by unclamping the inflow and outflow lines. The first 10 min of REP was conducted in Langendorff mode to avoid the development of fatal ventricular arrhythmias. Baseline parameters for each heart were registered following the 10 min of working perfusion before the induction of ISA. To examine the recovery of the left ventricle, cardiac function was assessed after 30, 60 and 120 min of REP. During the entire experimental procedure, HR was measured by a computer acquisition system . AF was measured by a calibrated flow meter. CF was assessed by the timed collection of the effluent dripping from the heart. CO was calculated as a sum of AF and CF. SV was generated as the ratio of CO and HR .w/v) solution of TTC in phosphate buffer (pH 7.4), and the samples were stored at −70 °C for subsequent analysis. The frozen samples were sectioned, weighted, and blotted dry. The dried sections were scanned on an Epson J232D flat-bed scanner . The infarcted area (white coloration) and the risk area (entire scanned section) were measured using planimetry software . Estimates of infarcted zone magnitude were subsequently obtained by multiplying infarcted areas by weight of each slice. The resulting numbers represent weight of the risk zone and the infarcted zone. IS was expressed as percentage of the weight of infarcted tissue and the weight of risk zone (whole heart).Estimations of infarcted area were conducted using the TTC staining method as previously described . Brieflyg at 4 °C for 10 min. The supernatant was transferred to a new tube and centrifuged at 8944× g at 4 °C for 20 min, and then the supernatant was used as a cytosolic fraction. Protein concentration was measured by a BCA Protein Assay Kit .Approximately 300 mg of cardiac tissues was homogenized using a Polytron homogenizer in isolating buffer  and centrifuged at 358× A total of 50 μg of protein in each sample was separated on TGX Stain-Free™ 12% acrylamide gels. Then, gels were exposed to UV light; thereby, trihalo compounds contained in stain-free gels covalently bind to tryptophan residues in proteins, allowing total protein quantification. After transferring the proteins to PVDF membranes for 1 h at 100 V, membranes were exposed to another brief irradiation, and the resulting fluorescence signals were recorded, and the signal intensity is proportional to the total protein volume. After blocking with 5% of non-fat dry milk in TBST, membranes were incubated with primary antibody solution at 4 °C overnight. HO-1 antibody was obtained from Abcam , 1:1000. The membranes were washed with TBST and incubated with HRP-conjugated secondary antibody solution. After washing, the membranes were incubated with Clarity Western ECL substrate  to visualize through enhanced chemiluminescence bands according to the recommended procedure . The chemiluminescent bands and each total protein lane intensity were measured by Image Lab 5.2.1 software . During quantification, protein density was measured directly on the membranes and reflected to total loaded proteins. Thus, this type of normalization eliminates the need to select housekeeping protein ,49.p < 0.05.Statistical analyses were performed using GraphPad Prism 7 software . The data are expressed as mean +/− SEM. One-way ANOVA followed by Dunnett’s post-hoc test was carried out for data analysis. Differences were considered to be significant at the value of In conclusion, our results suggest that iron produced by increased HO-1 activity in response to ISA/REP after long-term HD-BC treatment could have an important role in ISA/REP-induced cardiac injury. DFO, a hydrophilic IC, had only a mild effect on cardiac function and IS at the applied concentration in the case of hearts isolated from BC-treated rats; thus, higher dose(s) of DFO should be applied. Furthermore, additional experiments are necessary to clarify the precise role of HO-1 and iron in mediating the cardiovascular effects of BC. The modulation of the effects of iron related to HO-1 activity should be investigated by the application of other types of synthetic or natural iron chelators  and/or via the modulation of the keap1/Nrf2/HO-1 pathway by the application of the inhibitors of these signaling molecules. Furthermore, the inhibition of the ferroptotic signaling pathways could also be a good target for the modulation of the effects of iron originating from increased HO-1 activity as a consequence of HD-BC treatment. The precise role of BC under ischemia and reperfusion in connection with iron needs to be further elucidated.


An ecofriendly approach was developed for preparing copper-doped carbon dots (CDs) with superior photocatalysis using chromium-copper-arsenic (CCA)-treated wood waste as a precursor. Original wood (W-CDs), CCA-treated wood (C-CDs), and bioremediation CCA wood (Y-CDs) were used as the precursors. The chemical composition and structural, morphological, and optical properties, as well as the photocatalytic ability of the synthesized CDs varied with wood type. The C-CDs and W-CDs had similar characteristics: quasispherical in shape and with a diameter of 2 to 4.5 nm. However, the Y-CDs particles were irregular and stacked together, with a size of 1.5–3 nm. The presence of nitrogen prevented the formation of an aromatic structure for those CDs fabricated from bioremediation CCA wood. The three synthesized CDs showed a broad absorption peak at 260 nm and a weak absorption peak at 320 nm. Proof of the model study for the fabrication of luminescent CDs from CCA wood waste for bioimaging was provided. The degradation rate of CD photocatalytic MB was 97.8% for 30 min. Copper doping gives the CDs electron acceptor properties, improving their photocatalytic efficiency. This study provides novel ways to prepare nanomaterials from decommissioned wood as a nontoxic and low-cost alternative to fluorescent dots. Carbon nanomaterials, such as carbon nanodots (CDs) , carbon Nowadays, various synthesis approaches for CDs have been proposed, including acidic oxidation, electrochemistry, hydrothermal carbonization, discharge, ultrasonic synthesis, solvothermal synthesis, laser ablation, and microwave pyrolysis . HoweverCarbon nanodots have often been used for the photocatalytic degradation of organic pollutants . Like otFor sustainable development, an increasing number of natural precursors are recommended for fabricating CDs because of their abundance, ecofriendly character, and low cost. The reported natural precursors include actinidia deliciosa , soybeanWood utility poles are widely used in North America and Europe. They account for about 49.2% of the total global utility poles. Most wood utility poles are made from CCA-treated wood. CCA preservatives were initially designed to increase the resistance of the wood against microorganism decay in outdoor applications. However, owing to the high concentrations of toxic contaminants present in the CCA-treated wood and the increasingly large volumes of treated wood waste, it is urgent and important to find ways for the disposal of waste CCA wood . The CCAAlthough many decontamination approaches have been investigated to reduce the adverse effect of CCA-treated wood, recycling still attracts increasing attention. The upcycling of solid waste is environmentally friendly, cost-effective, and low energy, which meets the requirements of sustainable development . VariousCCA-treated wood waste has been directly thermally transformed into biochar, which has good dielectric properties , and theSaccharomyces cerevisiae was studied to prove the nontoxic properties of the obtained CDs for further bioimaging applications. The novel copper-doped carbon dots directly obtained from CCA-treated wood waste were considered a nontoxic and low-cost alternative to nano-photocatalysts and fluorescence nanodots. The CCA-treated wood waste was upcycled to fabricate fluorescent C-CDs without using any other industrial chemicals or metals. This approach reduced the environmental hazards caused by solid wood pollutants while achieving the high-value reuse of waste wood.This study explores obtaining CDs from CCA-treated wood via a hydrothermal carbonization method to explore their properties and utilization and compare them to that derived from the original wood. The synthesized CDs were characterized via high-resolution transmission electron microscopy (HR-TEM), X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD), Raman spectroscopy, and Fourier transform infrared (FTIR) spectroscopy to explore their properties and potential application. The chemical structure and morphological changes of the synthesized CDs based on various precursor wood materials were studied. Meanwhile, This study used three kinds of wood samples (Pinus koraiensis Sieb. et Zucc.) to prepare CDs, including original wood, CCA-treated wood, and bioremediation CCA wood. The obtained CDs were respectively labeled as W-CDs, C-CDs, and Y-CDs. A commercial wood served as the control group. Tred’si  supplied the waste CCA-treated wood. The Global Bioresource Center provided the Yarrowia lipolytica 20460. Saccharomyces cerevisiae was procured from Angel Yeast, Yichang, China.2SO4, KH2PO4 of 1.7 g, NaH2PO4 of 12 g, MgSO4·7H2O of 1.25 g, yeast extract of 0.5 g, and deionized water of 1 L.The mentioned bioremediation wood was the CCA wood treated with the Yarrowia lipolytica under the strain concentrations of OD = 0.6 for 15 days. In this case, the copper, chromium, and arsenic removal rate from the CCA-treated wood was 91.3%, 58.6%, and 32.8%, respectively. In brief, the strains were first cultivated in a yeast malt (YM) medium for 48 h. Then, the activated strains were transferred and cultivated in a growth medium in a shaker (200 mL medium/500 mL bottle). The YM medium was composed of yeast extract of 3 g, malt extract of 3 g, glucose anhydrous of 10 g, 5 g of tryptic soy broth, and 1 L of deionized water. The growth medium contained 50 g of glucose, 0.25 g of (NH4)The three kinds of wood samples were first grounded into powders. Then, 2 g of each sample was placed in a 100 mL Teflon-lined autoclave. Afterward, deionized water of 80 mL was added to each autoclave. The mixture was agitated with a glass rod, and then the autoclave was heated using an oven at 200 °C for 6 h based on a previous report . The CDs−1) were added to a solid YPD medium. Afterward, the activated strains were diluted 10,000 times, and 100 μL of the diluted strains was coated on the solid agar plates for cultivation for 24 h. The cellular toxicity experiment was performed in three replicates. The composition of the liquid YPD medium was mainly 1% yeast extract, 2% peptone, and 2% dextrose. The solid YPD medium was prepared by adding 2% agar to the liquid YPD medium. All mediums and materials were sterilized by high-pressure steam at 121 °C for 30 min before inoculation.In this experiment, Saccharomyces cerevisiae was added into 250 mL flasks containing a YPD liquid medium of 100 mL to activate the strains. All flasks were incubated at 30 °C on a rotary shaker with a speed of 120 rpm for 24 h. An assay was performed to compare the morphology and the numbers of Saccharomyces cerevisiae cells on YPD agar plates supplemented with or without the synthesized C-CDs (as an example). Specifically, the As-prepared C-CDs solutions of different concentrations  light. The power of the UV light used is 20 W, with a wavelength of 365 nm. The samples were collected after a set time and filtered through filter membranes of 0.22 μm. The absorbance value of the filtered supernatant was measured by a UV/vis spectrophotometer at 664 nm. All the experiments were performed in triplicate, and all results were marked as the mean ± standard deviation.A mixture of 0.1 g TiO−1. The phase structure of the CDs was characterized using an X-ray diffractometer  with Cu Kα radiation (λ = 1.5418 A) at 40 kV and 30 mA. The scanning speed was set as 20° min−1, and the 2θ ranged from 5 to 80°. Element analysis of the CDs was carried out using XPS equipment , and the Avantage software served to deconvolve the XPS spectra. XPS analyzed the As-prepared CD carbon, oxygen, nitrogen, copper, chromium, and arsenic elements. The fluorescence images were taken by a Leica fluorescence microscope , for which the samples were prepared by transferring the sole strain from the solid plate medium to a glass slide. CD Functional groups were recorded through FTIR, employing an IRTracer-100 with an ATR module . The analysis was performed with the spectra varying from 4000 to 500 cm−1 at a resolution of 4 cm−1 with an average of 64 scans.HR-TEM  analyzed the micromorphology of the obtained CDs. Sample analysis was performed by depositing the synthesized CD droplets on a carbon-coated Cu grid. When the Cu grid was dried at an ambient temperature, the CDs were coated on the Cu grid. Images were obtained with a 200 kV voltage and a beam current of 105 μA. A fluorescence spectrophotometer  was used to record the fluorescence spectra of the CDs. Spectra were acquired in a 1 cm quartz cuvette under the preset wavelength. The excitation wavelength varied from 260 to 400 nm. The excitation and emission slits were set to 5 nm with a voltage of 600 V. The Raman spectra were recorded with RENISHAW inVia spectrometer  at 532 nm, with a laser intensity of 0.05% and a wavelength range from 400 to 3200 cmY. lipolytica contain lots of heteroatoms and adhere to the CCA wood surface, which caused a difference in the forming process of the Y-CDs. The presence of nitrogen changed the molecular structure of the CDs, which has been reported before [The HR-TEM images presented in d before . The Y-Cd before . Howeverd before .–1, attributed to the O–H stretching vibration mode. The antisymmetric and symmetric stretching vibration C–H bands appeared at 2976 and 2898 cm−1, respectively [−1, the spectrum of the W-CDs and C-CDs appeared to show a characteristic peak at 1014 cm−1 for C–O stretching vibrations, whereas the Y-CDs were blue-shifted to 1057 cm−1 due to the existence of N elements [−1 corresponding to aromatic ring skeleton vibrational stretching, while the same absorption peak was not observed in the Y-CDs. It can be explained by the fact that the nitrogen doping prevented the formation of the aromatic ring skeleton in the Y-CDs. This can also explain the blue shift of the stretching vibration of carbon-oxygen bonds. The peaks appeared at 1670 and 1710 cm−1, corresponding to conjugated carbonyl and nonconjugated carbonyl stretching vibrations, respectively [−1 was weaker than that of the W-CDs and C-CDs, whereas the peak at 1710 cm−1 is the opposite. It was ascribed to the low aromatic ring content in the Y-CDs, reducing the conjugation effects. This observation agreed with previous reports that precursor materials impacted the production of CD functional groups [−1 corresponding to ether bonds and C–H bending were observed in the FTIR spectra, which is consistent with previous reports [The FTIR results indicated that various functional groups were present in the As-prepared CDs. As shown in ectively . Previouectively ,32. Withectively . The peal groups . Peaks a reports ,35. A pr reports . The obt reports . In addiAs shown in Another distinction was shown in the enlarged picture of N1s a, in whi2 carbons [All samples displayed the presence of four peaks in the C1 bands at around 284.6, 286.2, 287.8, and 288.8 eV, which were ascribed to C–C, C–O, C=O, and O–C=O groups, respectively b–d. The  carbons . The low3 orbitals, and G-bands are associated with sp2 carbon atoms in a two-dimensional hexagonal lattice [2 carbon atoms [Raman spectroscopy is commonly used to characterize carbon materials to confirm the form of the carbon atom. As shown in  lattice . The preon atoms . A possion atoms ,38. As son atoms . The broThe optical properties of the As-prepared CDs were analyzed in detail by ultraviolet (UV)-vis absorption and photoluminescence spectrometers. All the nanomaterials of the W-CDs, C-CDs, and Y-CDs revealed a broad absorption and a weak absorption peak in the UV band, respectively, located at 260 nm and 320 nm a. The prThe excitation spectra showed that the W-CDs, C-CDs, and Y-CDs had two peaks at 265 nm and 360 nm . SpecifiThe fluorescence intensity of the W-CDs, C-CDs, and Y-CDs increased first and then decreased with excitation wavelength, ranging from 310 nm to 400 nm. At the same time, the emission peaks were red shifted to longer wavelengths with increasing excitation wavelengths . The strTime-resolved luminescence was used to assess the lifetimes of the synthesized CDs b. The luSaccharomyces cerevisiae as the model strain in the current study. The number of strains that grew on the solid agar medium with 5–75 μL·mL−1 concentrations of C-CDs showed similar counts compared to the control sample without the CDs  [In the current study, although Cu ions were present in the synthesized C-CDs, they hardly affected the cell bioactivity of r cells) . All ther cells) . Subsequently, the bright blue fluorescence of the strains grown on the CD-doped medium was observed under a Leica fluorescence microscope. The strains in the control medium did not display fluorescence . The res2 composites were prepared for the further photocatalysis degradation of methylene blue (MB) dye. The absorption intensity of MB at 664 nm (characteristic peak) was recorded to analyze the dye degradation. 2 catalysts exited higher photocatalytic efficiency for MB dye degradation than the pure TiO2 catalysts, especially under visible light . TiO2 was commonly doped with other ions for the photocatalytic degradation of dyes [2 accepts the generated electrons from the CDs, thus transferring them to the dissolved oxygen in the solution. Under UV irradiation, the CDs transfer the electrons produced by TiO2, leading to the charge carrier recombination rate decreasing. Then, the electrons generate reactive oxygen radicals, and the holes contribute to the generation of -OH ions. The previous report also shows copper-doped carbon dots have great photocatalytic efficiency because of the increase in electron transfer rate and charge carrier lifetime [Photocatalytic degradation is another important application filed for CDs. However, the previous literature reports that pure CDs have no photocatalytic effect and can only be combined with semiconductors . CD/TiO2le light . This fi of dyes . The CDslifetime .2 and pure TiO2. The degradation rate of MB was about 97.8% after 30 min using the synthesized C-CDs under visible light. Thus, the synthesized C-CDs remained in nanoparticle states after the degradation of MB. The MB liquid, after photocatalysis, kept its fluorescence characteristics . The samples were characterized using the parameters of BET-specific surface area (SBET), external surface (Sext), total pore volume (VT), micropore volume (Vmi), and mesopore volume (Vme). Generally, a catalyst with a high specific surface area has great photocatalytic efficiency. In this study, the SBET of C-CDs/TiO2 (144.63 m2·g−1) was greater than that of the C-CDs (80.50 m2·g−1). However, the C-CDs have greater photocatalytic efficiency than C-CDs/TiO2. The reason may be that the C-CDs have a stronger light capture ability than C-CDs/TiO2 under visible light. Besides, the presence of Cu ions increased the electron transfer rate of the nano-photoactive catalysts, which is consistent with the previous literature [2 composites. Interestingly, the photocatalytic activity of the copper-doped C-CDs is higher than that of C-CDs/TiOeristics . The preThe solution proposed here applies to the recycling of all preservative wood waste. Due to the high number of wood products, such as wood utility poles, buildings, and railings, wood waste is a great potential carbon source for CDs. Recycling wood waste can slow carbon emissions and deforestation. The approach proposed here to prepare nanocarbon materials from wood is sustainable. The various applications of CDs have been explored in many studies, as shown in Saccharomyces cerevisiae, even with a high concentration of C-CDs in the medium. This confirms that they can be used as rare earth-free, ecofriendly, nontoxic, and low-cost phosphors for bioimaging. Heteroatom doping changes the structure and properties of carbon dots. For the Y-CDs, the nitrogen ions prevented the formation of the aromatic ring skeleton and decreased their size. Generally, pure carbon dots do not have a photocatalytic effect. However, due to the presence of copper, a strong photocatalytic effect in the reduction of MB was observed for the obtained C-CDs. Under indoor illumination, the CDs photocatalyzed the degradation of MB; the degradation rate was about 97.8% in 30 min. This observation reveals that the CDs directly obtained from CCA-treated wood waste can be used for photocatalytic wastewater treatment. The reported approach can extend to the reuse of wood waste as a sustainable resource, which provides a great potential way for producing metal-doped CDs via an ecofriendly and wide-ranging preparation method. Functional carbon nanodots have emerged as an urgent material due to their growing use in wide-ranging practical fields.In this work, novel copper-doped carbon nanodots were fabricated using hazardous CCA-treated wood waste as a carbon-containing precursor material. The synthesis pathway reported here is low-cost, ecofriendly, and organic solvent-free, making it possible for use in industrial manufacturing. The CDs obtained from the original wood, CCA-treated wood waste, and bioremediation CCA wood waste were characterized and compared. The obtained CDs were homogeneous with a similar average size of approximately 1.8–2.8 nm and a well-dispersed population. The XRD and Raman spectra results indicated that the CDs were predominantly amorphous at the macroscale. A blue emission of 440 nm was observed when the obtained C-CDs were under UV light. The cytotoxicity assay demonstrated that the obtained CDs had negligible effects on the growth of


We investigated the effects of lipolysis-stimulated lipoprotein receptor (LSR) on the tight junctions (TJs) of pancreatic ductal epithelial cells (PDECs) in hypertriglyceridemic acute pancreatitis (HTGAP).  Sprague-Dawley rats were fed standard rat chow or a high-fat diet and injected with sodium taurocholate to obtain normal and HTGAP rats, respectively. Serum triglyceride (TG) levels, pathological changes, TJ proteins in the pancreas, and TJ ultrastructure of PDECs were assessed. LSR overexpression (OE) and knockdown (KD) HPDE6-C7 models were designed and cultured in a high-fat environment. Protein levels were quantified by Western blotting. Cell monolayer permeability was detected using FITC-Dextran.  Serum TG concentration and pancreatic scores were higher in the HTGAP group than in the normal group. Among the TJ proteins, LSR protein expression was significantly lower in the HTGAP group than in the acute pancreatitis (AP) group. Tricellulin (TRIC) expression in the pancreatic ductal epithelia was higher in the HTGAP group than in the AP group. The HTGAP group had lower TJ protein levels, wider intercellular space, and widespread cellular necrosis with disappearance of cell junction structures. In the cell study, TJ proteins were downregulated and the cellular barrier was impaired by palmitic acid (PA), which was reversed by LSR-OE, whereas LSR-KD downregulated the TJ proteins and aggravated PA-induced cellular barrier impairment.  Hypertriglyceridemia downregulates the TJ proteins in PDECs, which may impair the pancreatic ductal mucosal barrier function. LSR regulation can change the effects of HTG on cellular barrier function by upregulating the TJ proteins. Hypertriglyceridemia (HTG) is the third most common etiology of acute pancreatitis (AP), following gallstones and alcohol. Patients with HTG suffer from severe AP (SAP) and organ failure . To date3− present in the pancreatic juice into the blood but also regulate the production of prosecretase in acinar cells [Pancreatic ductal epithelial cells (PDECs) and their secreted mucus layer constitute the pancreatic ductal mucosal barrier (PDMB), which can not only prevent the entry of bile and trypsin into the pancreatic parenchyma and the reflux of HCOar cells . The impar cells . Hence, The TJ is of two types: bicellular tight junction (bTJ) and tricellular tight junction (tTJ). The bTJ is mainly composed of occludin and claudin protein families. It controls the flow of macromolecules and ions into and out of cells. It plays an important role in the inflammation of the digestive tract, respiratory tract, and central nervous system, as well as in the invasion and metastasis of tumors . MoreoveThe obesity pandemic may be a likely contributor to the increasing incidence and worsening severity of AP , 11. ObeIn this study, we explored the effects of LSR on TJs in HTGAP using a rat model and a human pancreatic ductal epithelial cell line (HPDE6-C7) to provide a theoretical basis for the pathogenesis of HTGAP.In this study , we incln = 8), normal diet sham-operated group , normal diet AP group , high-fat diet control group , and high-fat diet AP group . Rats in the high-fat diet groups (HTG and HTGAP) were fed with a high-fat diet , and those in the normal diet group  were given standard rat chow.Rats were randomly divided into five groups: normal diet control group  into the biliary-pancreatic duct at a speed of 1 mL/min/kg. The pancreases of rats in the SO group were flipped and returned to the original position through laparotomy. Rats were anesthetized with intraperitoneal injection of 40 mg/kg pentobarbital sodium. Rats were euthanized by bloodletting at 24 h after modeling. The blood samples were collected from the abdominal aorta and centrifuged for 15 min at 2000 rpm, and the serum was conserved at -80°C. One part of the pancreas was immediately frozen in liquid nitrogen and conserved at -80°C for further analyses. The rest of the pancreatic tissue was fixed using 10% formalin, embedded in paraffin, and used for morphologic assessment by hematoxylin and eosin (H&E) staining.Triglycerides in the serum collected from the rats were detected using an Automatic Biochemistry Analyzer  following standard procedures.μm and stained with H&E staining. Two professional specialists, who were blinded to the experiments, morphologically evaluated the specimens under a light microscope. Changes in pancreatic histopathology were measured and classified in accordance with the study by van Laethem et al. [The pancreatic specimens of rats were sectioned at 3 m et al. .μL per slide; DAB, ZSGB-BIO, Beijing, China). The pathology image analysis system  was used for the visualization of these slides. The average of optical density (AOD) of images was measured using Image-Pro Plus 6.0 .Tissue sections collected from the pancreases of rats were deparaffinized, and antigens were retrieved through high pressure cooking with citrate buffer (pH 6.0) for 5 min. Then, these tissue sections underwent gradient cooling until room temperature and extensive washing in phosphate-buffered saline . Next, they were quenched with 3% hydrogen peroxide for 30 min, blocked with 3% bovine serum albumin at room temperature for 15 min, and incubated overnight at 4°C with 1 : 100 dilutions of the primary antibody against LSR , TRIC , claudin-1 , ZO-1 , occludin , and claudin-1 . The next day, the tissue sections were washed and incubated with a horseradish peroxidase-labeled secondary antibody  for 15 min at room temperature. The slides were placed in PBS, washed three times on a decolorizing shaker for 5 min per wash, and visualized with diaminobenzidine . The changes in TJs between the biliary and pancreatic ductal epithelia were observed using transmission electronic microscopy .2 at 37°C.The normal human pancreatic ductal epithelial cell line (HPDE6-C7) was purchased from the Beijing Beinachuanglian Biotechnology Research Institute  and cultured in an appropriate density with the recommended medium, supplemented with 10% fetal bovine serum in a humidified incubator containing 5% COAn LSR shRNA (targeting sequences of 5′-TGCTGAGCTACTCCTGTCAACGTCTCGTTTTGGCCACTGACTGACGAGACGTTCAGGAGTAGCT-3′) was cloned into a pcDNA6.2-GW/EmGFP-miR . This expression vector used plenti6.3/V5 DEST vectors. After amplification and DNA sequencing confirmation, this vector or negative control (NC) vector was used to generate lentiviruses in HEK-293 cells. These lentiviruses were used to infect HPDE6-C7 cells and named as LSR-knockdown (LSR-KD) cells or NC cells. In addition, a lentiviral vector carrying human LSR cDNA or vector only was used to infect HPDE6-C7 cells with LSR overexpression (LSR-OE) in cells. The efficiency of LSR-KD or LSR-OE was confirmed using RT-qPCR and Western blot. Moreover, to determine the effects of LSR on HPDE6-C7 cells in the HTG environment, cells were treated with 2 mM palmitic acid (PA)  for 24 h prior to use for subsequent experiments.μg) were separated using 10% SDS-PAGE and then transferred to a PVDF membrane. After blocking with 5% nonfat milk for 1 h, samples were incubated overnight at 4°C with primary antibodies against β-actin , LSR , TRIC , ZO-1 , occludin , and claudin-1 . Then, the membrane was washed three times using Tris-buffered saline with Tween-20 (TBST) buffer and incubated for 60 min at room temperature with peroxidase-conjugated AffiniPure Goat Anti-Rabbit secondary antibody . Protein bands were visualized using the Odyssey infrared laser imaging system  and were quantified through Quantity One software (Bio-Rad Laboratories).Cells were homogenized in RIPA lysis buffer , and protein concentrations were determined using the BCA protein assay kit . Total samples  was assessed through the passage of FITC-Dextran. Briefly, HPDE6-C7 cells were seeded into the upper chamber to form a confluent cell monolayer. Then, FITC-Dextran was added to the upper chamber. Finally, the medium at the bottom of the well was collected and the appearance of fluorescence was monitored at 490 nm excitation and 520 nm emission.t-test or analysis of variance (ANOVA) as appropriate, and the nonparametric variance test  was used to compare data with nonnormal distribution. One-way analysis of variance and the Bonferroni post hoc test were used to determine differences among multiple groups. Categorical variables were evaluated using the χ2 test, and Fisher's exact test was used when the number of observations was less than five. P < 0.05 indicated a statistical difference.All experiments were repeated at least three times and all data were expressed as mean ± standard deviation (mean ± SD), and categorical variables were expressed as mean (ratio). Data were analyzed using the statistical software SPSS 20.0 . Statistical significance was determined using Student's P < 0.05). Serum collected from the rats in the high-fat diet group was turbid, while that in the normal diet group was clear. The serum TG levels in rats in the HTG and HTGAP groups were significantly higher than the corresponding levels in the C group and AP group (715.88 ± 33.22 vs. 243.65 ± 23.26 μmol/L and 693.25 ± 35.97 vs. 263.70 ± 52.82 (P < 0.05), respectively)  .Edema, hemorrhage, and necrosis were observed in the macroscopic view of the pancreas in AP and HTGAP groups . FurtherThe expression levels of TJ proteins in the inflammatory and necrotic regions were decreased or disappeared. The expression levels of LSR in the HTG group were significantly lower than those in the C group. The expression levels of LSR, TRIC, occludin, ZO-1, claudin-1, and claudin-7 in AP and HTGAP groups were lower than those in the corresponding control group. The expression levels of LSR, TRIC, ZO-1, and claudin-1 in the HTGAP group were lower than those in the AP group. However, a significant difference was found in the LSR expression , Table 1In the HTG group, there were few cell-cell TJs in the biliary-pancreatic ductal epithelia, and the intercellular space was wide. However, the TJ count in the biliary-pancreatic ductal epithelial cells in the AP group was remarkably decreased, and the intercellular space in this group was wider than that in the group. Moreover, widespread cellular necrosis was observed in the HTGAP group. However, no cell-cell conjunctions were found in this group .In this study, we found that PA downregulated the expression levels of LSR, ZO-1, and claudin-1; upregulated the TRIC expression; and increased the monolayer permeability of HPDE6-C7 cells .LSR-OE upregulated the expression levels of ZO-1 and claudin-1, downregulated the expression levels of TRIC and occludin, and alleviated the cellular barrier impairment induced by PA. In turn, LSR-KD downregulated the expression levels of ZO-1 and claudin-1, upregulated the expression levels of TRIC and occludin, and aggravated the cellular barrier impairment induced by PA.In this study, we investigated the effects of LSR on TJs of PDECs in HTGAP. The findings of the study showed that the level of TG in the serum of rats in the high-fat diet group was significantly higher than that in the normal diet group. The rats in the high-fat diet group weighed significantly more than those in the normal diet group, and the increased weight in the high-fat diet group may have been a risk factor for HTGP. Obesity may contribute to increased AP incidence and worsened severity of AP by release of lipolytic UFA . In partκB [Our animal study showed that the pathological damage of the pancreas in the HTGAP group was more serious than that in the non-high-fat group. Animal studies have shown that the pathological damage of the pancreas in high-fat diet AP models is more serious than that in the non-high-fat AP model , 17. ThiκB  in acinaκB . StudiesκB  and inteκB , 17, 24 The TJ is a multifunctional macromolecular complex composed of a variety of proteins and molecules, which is widely present at the top of all connections between epithelial or endothelial cells and has a barrier function , 26. It TRIC is the first tTJ protein reported and named by Ikenouchi et al. in 2005 . TRIC isLSR is located on the cell membrane of the pancreatic duct in normal individuals and in well-differentiated pancreatic duct adenocarcinoma and is slightly expressed in the cytoplasm of poorly differentiated pancreatic duct adenocarcinoma cells . Currentβ, the expression of LSR can increase, and LSR and TRIC can be induced from tTJ positioning to bTJ [TRIC and LSR are major members of the tTJ and are expressed in various tissues. Studies have shown that TRIC is localized in normal human pancreatic acinar and ductal cells but not in pancreatic islet cells. Moreover, although TRIC is overexpressed in well-differentiated pancreatic duct adenocarcinoma, it is weakly expressed in poorly differentiated adenocarcinoma . In vitrg to bTJ . HoweverThe current limitation of our research concerns the interrelation of LSR and TRIC. PA can impair the intestinal barrier , 17, 24 In conclusion, HTG impairs the function of PDMB, probably by downregulating the TJ proteins in PDECs. Furthermore, the regulation of LSR may change the effect of HTG on the cell barrier function by regulating TJ proteins.


Pancreatic cancer (PAAD) is a malignant tumor with a poor prognosis and lacks sensitive biomarkers for diagnosis and targeted therapy. Cuproptosis, a recently proposed form of cell death based on cellular copper ion concentration, plays a key role in cancer biology. This study is aimed at constructing a risk model for predicting the prognosis of PAAD patients based on cuproptosis-related genes.  Pancreatic-related data from UCSC-TCGA and UCSC-GTEx databases were extracted for analysis, and TCGA-PAAD samples were randomly divided into the training and validation groups. Pearson correlation analysis was used to obtain cuproptosis-related genes coexpressed with 19 copper death genes. Univariate Cox and Lasso regression analyses were used to obtain cuproptosis-related prognostic genes. Multivariate Cox regression analysis was used to construct the final prognostic risk model. The risk score curve, Kaplan-Meier survival curves, and ROC curve were used to evaluate the predictive ability of the Cox risk model. Finally, the functional annotation of the risk model was obtained through enrichment analysis.  The Cox risk model has an eight prognostic cuproptosis-related gene signature. Kaplan-Meier survival curves demonstrated that the high-risk group had a shorter survival time. The ROC curve of the risk score was well created to predict one-, three-, and five-year survival rates, and AUC of the risk score was higher than other clinical characteristics. Cox regression analysis revealed that the risk score has an independent prognostic value for PAAD. GSEA reveals specific tumor pathways associated with the risk model .  We constructed a prognostic model containing eight cuproptosis-related genes  that can accurately predict the prognosis of PAAD patients. The results will provide new perspectives for individualized outcome prediction and new therapy development for PAAD patients. Pancreatic adenocarcinoma (PAAD) exhibits a poor prognosis in digestive system tumors because of its insidious onset, rapid development, and tendency to metastasize . Due to Copper is a basic element that is critical to the health of organisms . NormallLog Fold Change (LogFC) > 1 and p value < 0.05. Meanwhile, clinical information for all samples was downloaded, including age, sex, TNM stage, AJCC stage, overall survival (OS) time, progression-free survival (PFS) time, and disease-specific survival (DSS) time. Excluding samples with incomplete data, 178 patients were included.The Cancer Genome Atlas (TCGA) database contains RNA-seq data from 178 pancreatic and four normal pancreatic tissue samples . The GTEp value < 0.05.Tsvetkov et al., Deng et al., and Emami et al. proposed key genes involved in the tumor copper death mechanism, including ATP7A, ATP7B, CDKN2A, DBT, DLAT, DLD, DLST, FDX1, GCSH, GLS, LIAS, LIPT1, LIPT2, MTF1, NFE2L2, NLRP3, PDHA1, PDHB, and SLC31A1 , 12. Then = 88) and validation (n = 90) groups. The chi-squared test compared the clinical characteristics of patients in the training group and the validation group, aiming to evaluate the heterogeneity of the two sets of data. The data of the training group were used to construct the Cox risk model of cuproptosis-related genes. The cuproptosis-related genes were subjected to univariate Cox regression analysis based on OS, and the genes with prognostic significance were screened out (p < 0.01). To avoid overfitting the model, Least Absolute Shrinkage and Selection Operator (Lasso) regression analysis was performed on potential prognostic genes. When the cross-validation error of Lasso regression is the smallest, the list of prognostic genes corresponding to the best penalty parameter is obtained. Lasso prognostic genes were subjected to multivariate Cox regression analysis to determine the final risk model. The risk score of each patient was calculated by the following formula: risk score = βmRNA1 × ExpressionmRNA1 + βmRNA2 × ExpressionmRNA2 + ⋯+βmRNAn × ExpressionmRNAn.With R package “caret,” 178 cases were randomly assigned to the training  curve and the area under ROC curve (AUC) were employed to assess the predictive power of the risk score and each clinical characteristic. Finally, principal component analysis (PCA) was utilized to reduce the dimensions of four sets of genes  in the high- and low-risk groups, aiming to visualize the distinguishing ability of risk scores.n = 89) and the low-risk group (n = 89) of the entire cohort, and genes with absolute values of LogFC > 1 and p < 0.05 were defined as risk differential genes. Gene ontology (GO) can analyze biological processes (BP), cellular components (CC), and molecular functions (MF) of gene sets. The Kyoto encyclopedia of genes and genomes (KEGG) analysis is a systematic analysis of cellular pathways involved in genomes. GO and KEGG enrichment analyses were performed using “ClusterProfiler” and “ggplot2” packages in R (Version 3.6.3). Significant enrichment was considered eligible if p < 0.05. Gene set enrichment analysis (GSEA) can evaluate the regulation of pathways in the high- and low-risk groups. For GSEA in the entire cohort, GSEA software (GSEA 4.1.0) and h.all.v7.5.symbols were employed. A GMT dataset in MsigDB was used as the control group. The number of permutations was set to 1000. The screening criteria for significant enrichment pathways were false discovery rates (FDR) < 0.25 and absolute values of normalized enrichment score (NES) > 1. The nominal p value  was <0.05.Differential analysis was performed on the high-risk group  and human pancreatic cell lines were obtained from the Shanghai Institute of Nutrition and Health . RNA was isolated from samples using TRIzol, after which the PrimeScript RT Reagent Kit was employed to prepare cDNA. A StepOne Real-Time PCR Instrument  was used for all qRT-PCR analyses. Relative gene expression was analyzed using the 2-p < 0.05.Statistical analysis was performed using R (Version 3.6.3). Cox regression was used to evaluate each gene for the prognosis of PAAD patients by calculating the hazard ratio (HR) and its 95% confidence interval (CI). Log-rank tests were used to evaluate OS, DSS, and PFS of the three groups . The Pearson correlation test was used to analyze correlations. The ggplot2 R package was used to visualize all data. Statistical significance was set at n = 88) and validation (n = 90) groups. The clinical characteristics of 178 PAAD patients are detailed in The present study's flow diagram is displayed in p < 0.05 were identified as cuproptosis-related genes , and risk scores and risk groups for all patients are listed in Supplementary Table p < 0.001) (p < 0.001)   . The sam Figures  and the  Figures .DEGs between the high- and low-risk groups were used for functional enrichment analysis. We obtained 94 downregulated and 89 upregulated genes . GO and GSEA enrichment analysis was performed based on the high- and low-risk groups of the entire cohort samples, and eight signaling pathways that met the screening criteria were obtained Figures . Among tSurvival information and risk gene expression data of 107 PAAD samples were obtained by combining the GSE62452 and GSE28735 datasets. The risk scores of all patients were calculated based on the Cox risk model, and patients were divided into the high-risk and low-risk groups according to the best cutoff values. The risk scores for all samples used for external validation are shown in Supplementary Table Copper-dependent controlled cell death in human cells is a novel cell death mechanism different from the known cell death mechanism. It occurs through the direct binding of copper ions with lipacylated components of TAC in mitochondrial respiration. Cuproptosis leads to the aggregation of ribosylated proteins and subsequent downregulation of Fe-S cluster proteins, resulting in toxic protein stress and cell death . The fivThe current study constructed an eight-cuproptosis-related gene signature to predict OS, DSS, and PFS in PAAD patients. First, PAAD patients were randomly assigned to the training and validation groups. Based on the training group, prognostic cuproptosis-related genes were confirmed using Lasso regression and Cox regression models. According to the risk score, patients were categorized into the high- and low-risk groups. The prognoses between the high- and low-risk groups were statistically significant, and AUC suggests the prediction ability of our risk score signature. More importantly, by comparing ROC curves of patient survival, we found that the risk score based on cuproptosis-related genes had more accurate predictive power than clinicopathological indicators as follow-up years increased. The model was validated in the validation and entire groups. In addition, the model was externally validated using the GEO database, and the results showed similar predictive power. We refer to other cell death-based risk models for comparison with the cuproptosis risk model. The AUC values of the risk model in our study led to the predictive performance of overall survival at 1, 3, and 5 years, reaching 0.75, 0.84, and 0.86, respectively. In other cell death-related studies, the AUC values for overall survival at 1, 3, and 5 years were 0.537, 0.731, and 0.852 for autophagy ; 0.665, Our risk score included eight cuproptosis-related genes: AKR1B10, KLHL29, PROM2, PIP5K1C, KIF18B, AMIGO2, MRPL, and PI4KB. As a carcinogenic protein, AKR1B10 promotes tumor occurrence and development by enhancing fat production and is involved in pancreatic carcinogenesis via modulating the Kras-E-cadherin pathway , 25. PROα [We performed an enrichment analysis based on the risk scores of pancreatic cancer patients. GSEA revealed that various signaling pathways involved in cancer occurrence and development are enhanced in the high-risk group. The Myc oncoprotein family is involved in regulating metabolic reprogramming and providing sufficient energy for cancer cell proliferation . Aerobicα . mTORC1 α . The E2Fα . Similarα . In addiα .This study has limitations. First, some incomplete information was deleted, which led to a certain degree of information deviation. Next, the concept of cuproptosis was only recently proposed, and sufficient literature cannot be obtained to clarify the possible mechanism of an eight-gene signature involved in cuproptosis. Multicenter, large-scale clinical trials of prediction models require further validation.In conclusion, we constructed a prognostic model containing eight cuproptosis-related genes and analyzed their MF in PAAD. Our study highlights that the risk score we constructed can be used as a marker of outcome in PAAD patients. Our findings provide new insights into the role of cuproptosis-related genes in PAAD pathogenesis and as potential biomarkers for PAAD diagnosis and treatment.